Several methods for the detection of biomolecules, in particular of low concentration in a liquid, are known in the art. One method is based on so-called evanescent waves. The biomolecules to be detected are in a liquid solution and are brought into close contact with the surface of a medium, (e.g. glass), which is optically denser than the liquid used. This may be performed by adsorption of the biomolecules to complementary biomolecules immobilized on the glass surface. The biomolecules to be detected are marked with a fluorescent compound. Such may either be performed directly (i.e., by means of chemical binding between the biomolecule and the fluorescent substance), or in that biomolecules labelled with a fluorescent dye and immobilized or chemically bound to a part of the detection apparatus compete with the biomolecules to be detected; i.e., the unknown biomolecule releases a fluorescent biomolecule, which in turn adsorbs to the complementary biomolecule immobilized on the glass surface. The latter process is described in Badley, R. A., et al., "Optical Biosensors For Immunoassays: The Fluorescence Capillary-fill Device", Phil. Trans. R. Soc. Lond. B 316 (1987), pp. 143-160.
Monochromatic light, from a laser source or a filtered flash lamp, strikes the boundary surface between the optically denser medium (e.g., glass) and the optically rarer medium (e.g., an aqueous solution). The light beam is incident from the optically denser medium, and the angle of incidence is equal to or larger than the critical angle so that total internal reflection occurs. When such happens, an evanescent wave is created in the optically rarer medium (aqueous solution); this evanescent wave penetrates a fraction of a wavelength into the optically rarer medium. The electric field amplitude of the evanescent wave is largest at the boundary surface and decays exponentially with the distance from the interface.
Due to the limited depth of penetration of the evanescent wave, such wave is suited to monitor the presence of biomolecules at the boundary surface. It causes the fluorescent appendix of the biomolecules to emit light of a wavelength longer than the incident wavelength (this is effectively how fluorescence is defined). The fluorescence signal can be measured directly by monitoring the scattered light, or by measuring the light coupled back into the optically denser medium.
It is understood that the above technique is not limited to adsorption to a glass/aqueous solution interface. Instead, other materials may be used as well. It is further possible to use other effects than fluorescence which shifts the wavelength of the incident light to larger wavelengths, (e.g., phosphorescence or absorbance). In the latter case, even unlabelled biomolecules may be detected. The general method of detection is based on measuring a refractive index change caused by the presence of the biomolecules monitored by the refracted or reflected light, or, in other words, by the deviation of the angle of the reflected or refracted light. It is also known in the art to direct the incident light such that it is reflected multiple times in a waveguide, so that it strikes the boundary surface multiple times, see e.g., Sutherland, Ranald M. et al., "Optical Detection of Antibody-Antigen Reactions at a Glass-Liquid Interface", Clin. Chem. 30/9 (1984), p. 1533-1538.
Another technique for the detection of biomolecules is based on so-called surface plasmon resonance (SPR). This method requires a thin metal film, layer or coating (in more general terms, a conductive or semiconductive layer) between the glass and the liquid solution. Incident light, if impinging at a certain angle, causes surface modes (TE and/or TM modes) associated with collective electron oscillations to propagate along the interface between the metal film and the optically rarer medium (e.g., liquid solution). The incident light is usually coupled into the metal film by means of a prism or a grating. At a specific wavelength or angle, resonance occurs resulting in a sharp minimum or dip of reflectivity. The resonance condition is dependent upon the optical characteristics of the metal film, its thickness, the refractive indices of the dielectrics on either side of it (if any) and the angle of the incident light.
The first two of these characteristics remain basically unchanged in a given apparatus for performing surface plasmon resonance. However, the refractive index of the optically rarer medium varies with the amount of biomolecules bound or adsorbed to its surface. This is the property to be monitored.
In order to detect the presence and the amount of adsorbed biomolecules, either the variation in reflectivity at a given angle of incident light may be monitored, or the resonance shift (the reflectivity minimum is shifted to a different angle of incidence upon the presence of biomolecules) may be observed.
Surface plasmon resonance may be caused either by a metal grating, or by an evanescent wave resulting from total internal reflection (see above).
For further details of the surface plasmon resonance technique, reference is particularly made to Daniels, P. B. at al., "Surface Plasmon Resonance applied to Immunosensors, Sensors and Actuators", 15 (1988), p 11-18, and Kooyman, R. P. H. et al., "Surface Plasmon Resonance Immunosensors: Sensitivity Considerations", Analytica Chimica Acta, 213 (1988), p. 35-45.
Prior art surface plasmon techniques used the change of the refractive index caused by the biomolecules in order to detect their presence and/or their concentration. However, it is also known in the art to use the evanescent wave associated with surface plasmons to excite fluorescence or phosphorescence in an immunoassay, as described in EP-A-353 937.
A general problem in applying surface plasmon resonance is that the biomolecules may be "overexcited", i.e. too much energy is transmitted from the incident electromagnetic wave to the biomolecules. In such case, the biomolecules may bleach out, i.e., they alter their characteristics and their physical behavior, such that they may be no longer detectable. It will be appreciated that this effect impairs the results of the measurement.
The problem is that there is no control of the amount of energy "pumped" into the biomolecules. Of course, the energy emitted by the source of p-polarized electromagnetic waves is known and may be varied. However, the fraction of the total emitted energy pumped into the biomolecules is unknown (i.e., although the total emitted energy is known, the portion thereof passed on to the biomolecules is unknown). The reason is that, in prior art arrangements for the detection of biomolecules, the angle of incidence is not the exact angle at which surface plasmon resonance occurs (this angle will be called .theta..sub.SPR hereinafter) either because this angle cannot exactly be met, or, even if it were possible to adjust the apparatus exactly to .theta..sub.SPR, this angle would be subject to drift caused by temperature effects, binding of unspecific molecules (i.e., molecules not subject to detection), changes at the metal/liquid interface, etc. Therefore, even the latter approach would lead to some deviation of the actual angle of incidence, with respect to the optimum angle .theta..sub.SPR for surface plasmon resonance.
The consequence of the actual angle of incidence not exactly corresponding to the optimum angle for surface plasmon resonance is that only a fraction of the total energy carried by the p-polarized electromagnetic waves is passed on to the biomolecules. On the other hand, and as outlined above, this fraction is unknown. Even if it were possible to determine the deviation of the actual angle versus the ideal angle precisely, such would not solve the problem, as the fraction of energy coupled into the biomolecules is not a well-defined function of the deviation.
Therefore, in prior art arrangements, effective control of energy coupled into the biomolecules has been substantially impossible. It is, of course, possible to reduce the total amount of energy emitted by the radiation source such that even a 100% energy coupling would not damage the biomolecules. However, assume that a deviation between the actual angle of incidence and the ideal angle .theta..sub.SPR for surface plasmon resonance causes a reduction of energy transmission of 75%. In such a case, the remaining energy pumped into the biomolecules would be insufficient for reliable measurements. On the other hand, if the total energy output of the radiation source were substantially increased, operation could be performed reliably even upon a very little energy coupling ratio, but a 100% coupling would then damage the biomolecules.